This invention relates to methods for assay of epidermal cell growth factors, in particular factors affecting keratinocyte growth and migration.
In general growth factors are chemical compounds which promote or stimulate the growth of mammalian cells in vitro and/or in vivo. Examples include epidermal growth factors (EGF's) such as the polypeptide of molecular weight 6,045 reported by Savage et al., 1972, 247 J. Biol. Chem 7612, and the .alpha. transforming growth factor (TGF-.alpha.) reported by Anzano et al., 1983, 80 Proc. Nat. Acad. Sci. 6264.
Rheinwald et al., 1977, 265 Nature 421, discloses inoculating 5.times.10.sup.3 epidermal keratinocytes into a culture dish containing lethally irradiated 3T3 cells and varying amounts of EGF. The presence of EGF reportedly did not affect cell growth rate but allowed longer periods of exponential growth and resulted in larger and thinner colonies (approximately 7 mm.sup.2 in surface area).
Another assay, disclosed by Smith et al. (1985, 315 Nature 515), involves injection of growth factors into newborn mice and measuring the time to eyelid opening. Growth factors such as EGF and TGF-.alpha. accelerate eyelid opening.
Anzano et al., supra, describe the formation of approximately 3,000 colonies (7,000-15,000 .mu.m.sup.2 in surface area) from NRK clone 49 F cells grown for 7 days in soft agar in the presence of TGF-.alpha. and TGF-.beta.. They characterize such colonies as large, and rely on them as an assay for growth factors.
Keratinocytes are the principal cell type in mammalian epidermis. They can be obtained by disaggregating epidermal sections with trypsin and EDTA, as described by Rheinwald et al. (1977, 265 Nature 421). In cell culture they depend on the support of fibroblasts, such as 3T3 cells, in order to initiate colony formation. Appropriate culture media are described by Simon et al. (1985, 40 Cell 677) and Rheinwald et al., supra.